Table1_Interleukin-1 receptor-dependent and -independent caspase-1 activity in retinal cells mediated by receptor interacting protein 2.DOCX

IntroductionInflammation and cell death play an important role in the pathogenesis of diabetic retinopathy. Previously we observed sustained activation of pro-inflammatory caspase-1 in retinas of diabetic animals and patients. In this study, we aimed to look at mechanisms underlying chronic caspase-1 activation in vitro and in vivo.MethodsNon-diabetic and diabetic wild type and IL-1 receptor (IL-1R1) knockout mice were used for in vivo experiments. Diabetes was induced using STZ (streptozotocin). Human Müller cells were used for in vitro studies. Cells were treated with either 5 mM or 25 mM glucose or interleukin-1beta (IL-1β) in the presence or absence of IL-1 receptor antagonist (IL-1ra) or siRNA against RIP2 (receptor interacting protein-2) for up to 96 h. Outcome measurements to assess Müller cell functions included measurements of caspase-1 activity using a fluorescence peptide substrate, production of IL-1β by Elisa, and cell death using trypan blue exclusion assays.ResultsOur in vivo results demonstrate that caspase-1 activation progresses from an IL-1R1 independent mechanism at 10 weeks of diabetes to an IL-1R1 dependent mechanism at 20 weeks indicating that feedback through IL-1R1 is crucial for sustained caspase-1 activity in retinas of mice. A similar hyperglycemia-mediated caspase-1/IL-1β/IL-1R1 feedback signaling was detected in vitro in human Müller cells which was prevented by treatment with IL-1ra. Our data also indicate that hyperglycemia induces caspase-1 activation initially but IL-1β sustains caspase-1 activation via caspase-1/IL-1β/IL-1R1 feedback and we identified RIP2 as mediator for both hyperglycemia- and IL-1β-induced caspase-1 activation. Activation of caspase-1/IL-1β/IL-1R1 feedback signaling caused Müller cell death which was prevented by RIP2 knockdown.DiscussionWe conclude that any intervention in caspase-1/IL-1β/IL-1R1 feedback signaling presents novel therapeutic options for the treatment of diabetic retinopathy.

本研究引入了糖尿病视网膜病变的发病机制中炎症和细胞死亡的重要作用。先前研究表明，糖尿病动物和患者的视网膜中持续存在促炎型caspase-1的激活。本研究旨在探究体外及体内慢性caspase-1激活的潜在机制。研究方法中，我们使用了非糖尿病及糖尿病野生型和小鼠IL-1受体（IL-1R1）敲除小鼠进行体内实验，利用STZ（链脲佐菌素）诱导糖尿病。体外研究中，我们使用了人类Müller细胞。细胞处理方案包括5 mM或25 mM葡萄糖或白介素-1β（IL-1β）的存在或不存在下，在IL-1受体拮抗剂（IL-1ra）或针对RIP2（受体相互作用蛋白-2）的siRNA存在或不存在下，持续处理至96 h。评估Müller细胞功能的指标包括使用荧光肽底物测量caspase-1活性、通过Elisa检测IL-1β的产生以及使用台盼蓝排除法检测细胞死亡。结果显示，体内实验中，caspase-1的激活从糖尿病10周时的IL-1R1非依赖机制进展至20周时的IL-1R1依赖机制，表明通过IL-1R1的反馈对于维持小鼠视网膜中caspase-1的持续激活至关重要。体外实验中，在人Müller细胞中也检测到了由高血糖介导的caspase-1/IL-1β/IL-1R1反馈信号，且该信号可通过IL-1ra治疗得到阻断。数据还表明，高血糖最初诱导caspase-1激活，但IL-1β通过caspase-1/IL-1β/IL-1R1反馈途径维持caspase-1的激活，我们鉴定RIP2为高血糖和IL-1β诱导的caspase-1激活的介质。caspase-1/IL-1β/IL-1R1反馈信号的激活导致Müller细胞死亡，而RIP2敲低可以防止细胞死亡。讨论部分，我们得出结论，针对caspase-1/IL-1β/IL-1R1反馈信号进行的任何干预都为糖尿病视网膜病变的治疗提供了新的治疗策略。